Summary: Spinal muscular atrophy (SMA) is a devastating genetic disease of infants and children. In >95% of cases, the disease results from the loss of Survival Motor Neuron 1 (SMN1), the genetic source of survival motor neuron (SMN) protein. A nearly identical paralog gene, SMN2, cannot compensate for SMN1 loss due to a C to T mutation at position 6 (C6U in the transcript) in exon 7. C6U causes predominant exon 7 skipping and the resulting transcript produces a truncated protein that is rapidly degraded. Since most SMA patients carry at least one copy of SMN2, a vast majority of therapeutic approaches employ SMN2 as a target to increase the full-length transcript and SMN protein. One of the most promising means to increase SMN2 exon 7 inclusion is by antisense oligonucleotides (ASO) to block negative elements that contribute to exon 7 skipping. ASOs targeting intronic splicing silencer- N1 (ISS-N1) have shown remarkable efficacy in preclinical and clinical studies. This proposal is inspired by several new developments, including the availability of newly identified targets within SMN2 intron 7 and success of a dual-masking ASO that has shown better efficacy than ASOs against ISS-N1 or other individual targets. A dual-masking ASO is a single oligonucleotide molecule that simultaneously sequesters two targets often separated from each other by hundreds of nucleotides. The most plausible mechanism of action of a dual-masking ASO is structural rearrangement within SMN2 intron 7. We have recently solved the structure of SMN2 intron 7 and have experimentally validated an unusual structure-associated regulatory element that exerts its inhibitory effect through a unique long-distance interaction. In this proposal, we will take advantage of several structure- associated regulatory sequences within SMN2 intron 7 to design novel dual-masking ASOs for an efficient splicing correction in SMA. In Aim 1, we will construct a library of dual-masking ASOs employing seven novel target combinations. We will test the efficacy of dual-masking ASOs in SMA patient fibroblasts for their ability to restore SMN2 exon 7 inclusion. Based on prior successes, we will perform all initial screenings with oligonucleotides carrying phosphorothioate backbone and 2-O-methyl modifications (2OMe). We will employ various assays to select efficient dual-masking ASOs that show high splicing correcting capability at low concentrations (5 nM to 50 nM). We will test the specificity of dual-masking ASOs employing appropriate mismatch controls. We will use an ISS-N1-targeting ASO as an additional control. For up to three top performing dual-masking ASOs, we will determine the levels of SMN and other proteins that are likely to be upregulated in response to the increased expression of SMN. In Aim 2, we will employ a mild (allele C) and a severe (D7) mouse model to determine the therapeutic efficacy of the lead dual-masking ASO identified in Aim 1. We will first perform short-term pilot studies to determine the efficacy of different ASO doses and modes of delivery. We will perform subsequent long-term studies at an optimized dose and route of delivery. Upon administration of the lead ASO in allele C mice, we will monitor body weight, tail and peripheral necrosis, cardiovascular function (employing electrocardiography) and nociception (employing mechanical and thermal sensitivity tests). We will perform histology on various tissues of allele C mice to monitor the effect of ASO treatment. We will evaluate the expression of genes known to be impacted by low SMN in brain, spinal cord and testis of allele C mice. Upon administration of the lead ASO in D7 mice, we will monitor lifespan, body weight and peripheral necrosis. We will evaluate motor, muscle and cardiovascular functions of D7 mice. We will examine histology of muscle, spinal cord and neuromuscular junctions in D7 mice to monitor the effect of ASO treatment. We will perform RNA-seq in samples collected at P7 and P15 to examine early and late effects of ASO treatment on the transcriptome in brain and spinal cord of D7 mice. The outcomes from this proposal will likely produce an efficient drug for a potential therapy for SMA.